EP0012444A1 - Diagnostic method, especially for the early recognition of malignant tumors and/or viral diseases, and means to carry it out - Google Patents

Abstract

1. A diagnostic method, in particular for the early detection of malignant tumors and/or viral diseases in the human or animal organism, using a body fluid collected from the organism being examined for the content of specific pteridines, characterized in that 7,8-dihydropteridine or its oxidation products or its reduction products or their substituted products, with the exception of biopterin and its reduction products, is qualitatively detected and/or semi-quantitatively or quantitatively determined in the body fluid.

Description

The invention relates to a method for diagnosis, in particular early detection of malignant tumors and / or viral diseases in the human or animal organism, wherein a body fluid taken from the organism is examined for the content of certain pteridines, and means for carrying out the method.

Today, malignant tumors are diagnosed almost exclusively on the basis of clinical, radiological, endoscopic, histological and cytological examinations. To date, no clinical chemical or biochemical findings that allow a reliable prediction of a malignant tumor exist. The quantitative determination of these hormones in serum or urine can provide important diagnostic information, particularly in the case of endocrine disruptors that produce more hormones. However, the increased hormone levels do not indicate malignant neoplasms, but can also occur in other diseases.

More recently, the determination of some tumor-associated antigens, e.g. B. the carcinoembryonic antigen (CEA) or the α ₁ -Fetoproteins attained a certain importance. Since these antigens are increased, depending on the tumor, in the best case only in 30 - 90% of the tumor patients, and, on the other hand, elevated values are also found in patients with other diseases, the practical value of these studies remains very limited.

A tumor diagnosis based on the determination of the content of certain pteridines in body fluids appears to be more promising, although not yet used in practice. Kokolis and Ziegler (Kokolis N. and Ziegler J., Cancer Biochem. Biophys. 1977, Vol. 2, pp. 79-85) found elevated levels of tetrahydrobiopterin in the serum of tumor patients. Studies by Halpern et al. (Cancer Research 38, 2378-2384, 1978,) revealed increased amounts of 6-hydroxymethylpterine in tissue cultures of malignant cells after prior folic acid administration.

It has now been found that, surprisingly, increased amounts of certain pteridines are also present in viral diseases and can be detected.

• 1) Isolierung, Züchtung und Identifizierung des Virus vom Kranken (Dauer.der Untersuchung ca. 6 Wochen);
• 2) Nachweis von virusspezifischen Antikörpern im Serum oder Liquor des Kranken (Dauer der Untersuchung ca. 3 Wochen);
• 3) Züchtung und Identifizierung von Bakterien des Kranken, bei negativem Ergebnis Ausschluß der bakteriellen Erkrankung und Annahme einer viralen Erkrankung (indirekter Nachweis - Dauer der Untersuchung 2 bis 3 Tage, Wiederholungsuntersuchungen erforderlich).

Clinical laboratory diagnosis of viral diseases is currently essentially based on three procedural principles:

• 1) Isolation, cultivation and identification of the virus from the patient (duration of the examination approx. 6 weeks);
• 2) detection of virus-specific antibodies in the patient's serum or cerebrospinal fluid (duration of the examination approx. 3 weeks);
• 3) Cultivation and identification of bacteria from the patient, if the result is negative, exclusion of the bacterial disease and acceptance of a viral disease (indirect detection - duration of the examination 2 to 3 days, repeat examinations required).

The methods mentioned under point 1, which are also referred to as "large virus diagnostics", are costly and tedious. They are also not always satisfactory for the clinic; this applies e.g. B. when diseases of the upper airways or meningoencephalitic syndromes are to be clarified. In these situations, isolation and identification mean no diagnostic help for the individual case, because their results can be read too late.

• a) Der Neutralisationstest
• b) Der Hämagglutinations-Hemmungstest (Hirst-Test)
• c) Die Komplementbindungsreaktion (KBR).

The methods mentioned under point 2 are easier to carry out and of greater importance for the operation of the clinic.

• a) The neutralization test
• b) The Hemagglutination Inhibition Test (Hirst Test)
• c) The complement fixation reaction (KBR).

All of these studies require that they be performed quantitatively on two serum samples from the same patient at the same time. The first serum sample should be taken immediately after the clinical symptoms appear, the second serum sample after about 14 days. The serum samples are sent in immediately after collection and frozen in the laboratory. The serum pair is always examined on the same day. This "small virus diagnosis" also results in a significant time factor and considerable diagnostic and costly effort.

The third method is an exclusion proceed with all defects inherent in these processes. The viral disease itself is not directly proven.

The object of the invention is to provide an improved clinical chemical method for diagnosis, in particular early detection of malignant tumors and / or viral diseases, and for monitoring the course of the diseases in human or animal organisms, by determining certain pteridines in body fluids.

This is achieved according to the invention in that a preferably unconjugated, optionally derivatized 7,8-dihydropteridine or its oxidation product or its reduction product is qualitatively detected and / or semi-quantitative or quantitative in the body fluid is determined.

The 7.8 dihydropteridine has the following formula:

das 7,8-Dihydrolumazin

das 7,8-Dihydroxanthopterin

die 7,8-Dihydro-7-xanthopterincarbonsäure

das 7,8-Dihydro-isoxanthopterin

die 7,8-Dihydro-6-isoxanthopterincarbonsäure

Examples of dihydropteridines are: 7,8-dihydropterin

the 7,8-dihydrolumazine

the 7,8-dihydroxy hopterin

the 7,8-dihydro-7-xanthopterin carboxylic acid

the 7,8-dihydro-isoxane hopterin

the 7,8-dihydro-6-isoxanthopterin carboxylic acid

The previous examples concerned unconjugated pteridines. Folic acid is an example of a conjugated pteridine:

The inventive method is based on the knowledge gained in animal experiments that, for. B. in the urine of Ehrlich ascites tumor-bearing or virus-infected mice a significantly increased excretion of 7,8-dihydro-6-hydroxylumazine, a 7,8-dihydropteridine occurs, which can be detected very early and very well with tumor growth or with the severity of the infection of the model animals correlated. It has also been found in humans that the (increased) occurrence of 7,8-dihydropteridines in body fluids is a reliable indication of malignant growth and / or viral diseases.

In addition, the method can be better adapted to the special circumstances in humans by using neopterin (6-erythro-1,2,3 trihydroxypropyl-pterine) or its hydrogenated (reduced) or substituted product in the body fluid, preferably in the urine is qualitatively proven and / or determined semi-quantitatively or quantitatively.

• als D-Neopterin
  
  als L-Neopterin
  
  bzw. als Racemat des Neopterins. Als reduziertes Derivat des _Neopterins kommt insbesondere das Biopterin mit der Formel
  
  in Frage.

The neopterin to be detected according to the invention can be present in the following way:

• as a D-neopter
  
  as L-Neopterin
  
  or as a racemate of neopterin. As a reduced derivative of _N eopterins particular the biopterin is of the formula
  
  in question.

The dihydropteridine to be detected according to the invention can be substituted in different ways, for example by groups such as OH, NH ₂ , CH ₃ , CH ₂ OH, COOH, CHO, alcohols or sugar.

The neopterin to be detected according to the invention can be substituted in different ways, for example by sugar or phosphate. The dihydropteridine, the neopterin or its hydrogenated or substituted product can be found in various body fluids, e.g. B. blood, serum, urine, saliva u. a. be detected. The method according to the invention does not necessarily presuppose that a pteridine derivative or a preparation interfering with the pteridine metabolism is administered beforehand to the organism from which the body fluid to be examined is removed.

• - Niedervolt- oder Hochspannungselektrophorese auf Trägern oder trägerfrei kombiniert mit in-situ-Fluorometrie bzw. Remissionsmessung im UV- bzw. sichtbaren Bereich nach Anfärbung;
• - Dünnschichtchromatographie kombiniert mit in-situ-Fluorometrie oder Remissionsmessung im UV- bzw. sichtbaren Bereich nach Anfärbung;
• - Papierchromatographie kombiniert mit in-situ-Fluorometrie oder Remissionsmessung im UV- bzw. sichtbaren Bereich nach Anfärbung;
• - Gaschromatographie;
• - Gaschromatographie kombiniert mit Massenspektrometrie;
• - Felddesorptionsmassenspektrometrie;
• - Hochdruckflüssigkeitschromatographie und Fluoreszenzdetektion bzw. Detektion im UV- bzw. sichtbaren Bereich nach chemischer Reaktion bzw. elektrochemische Detektion;
• - Hochdruckflüssigkeitschromatographie und Massenspektro-f metrie;
• - Flüssigchromatographie an Ionenaustauschern;
• - Photometrie;
• - Fluorometrie;
• - Spektroskopie (UV-, IR-, kernmagnetische Resonanz, Massenspektrometrie);
• - Immunologische Methoden (Radio-Immuno-Assay (RIA), Enzyme-Linked-Immuno-Sorbent-Assay (ELISA), Enzym-Immuno-Assay (EMIT), Immunelektrophorese u. a.);
• - Isotopenverdünnung;
• - Enzymtest, z. B. mittels Xanthinoxidase;
• - Farbtest;
• - Biologischer Test (Crithidia-Wachstumstest u. a.);
• - iso-elektrische Fokussierung;
• - kinetische Nephelometrie.

The qualitative detection of a 7,8-dihydropteridine or neopterin or its oxidation products, reduction products or substituted products is sufficient if the healthy organism in question does not contain 7,8-dihydropteridine in the body fluid to be examined, ie without the presence of a malignant tumor or a viral disease delivers. Otherwise, a quantitative or semi-quantitative determination of the 7,8-dihydropteridine or neopterin or its oxidation products, reduction products or substituted products is necessary, whereby these values are to be compared with normal values (of healthy people) depending on the specific examination method of the body fluid. Conventional analytical methods are suitable for the detection or quantitative determination of the pteridine derivatives according to the invention or of neopterin, including the oxidized, hydrogenated or substituted products, such as:

• - Low-voltage or high-voltage electrophoresis on carriers or carrier-free combined with in-situ fluorometry or reflectance measurement in the UV or visible range after staining;
• - Thin layer chromatography combined with in-situ fluorometry or reflectance measurement in the UV or visible range after staining;
• - Paper chromatography combined with in-situ fluorometry or reflectance measurement in the UV or visible range after staining;
• - gas chromatography;
• - gas chromatography combined with mass spectrometry;
• - field desorption mass spectrometry;
• - High pressure liquid chromatography and fluorescence detection or detection in the UV or visible range after chemical reaction or electrochemical detection;
• - high pressure liquid chromatography and mass spectrometry;
• - liquid chromatography on ion exchangers;
• - photometry;
• - fluorometry;
• - Spectroscopy (UV, IR, nuclear magnetic resonance, mass spectrometry);
• - Immunological methods (radio immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EMIT), immunoelectrophoresis, etc.);
• - isotope dilution;
• - enzyme test, e.g. B. by means of xanthine oxidase;
• - color test;
• - Biological test (Crithidia growth test etc.);
• - iso-electric focusing;
• - kinetic nephelometry.

Preferred in practice, because on the one hand very specific, on the other hand, the immunological methods such as RIA, ELISA and EMIT can be used in routine operation with relatively simple means.

For this it is necessary to obtain antibodies against the pteridines to be determined, for example xanthopterin, neopterin or biopterin. These antibodies are obtained in a manner known per se by immunizing experimental animals, preferably rabbits, with suitable pteridine conjugates. The coupling of the pteridines to the immunogenic carriers, e.g. B. serum albumin, is carried out according to methods known per se. The required specificity of the antibodies for the particular pteridine to be determined is obtained by the selection of the pteridine derivatives used for the coupling.

Antibodies for pterins generally substituted in the 6-position by a side chain are obtained by coupling the hapten to the immunogenic carrier via the 6-position.

Here, for. B. Xanthopterin with ethylene glycol under acidic catalysis and then oxidized the terminal OH group to the carboxy group. This reactive hapten derivative is with a carbodiimide in a conventional manner, for. B. conjugated to bovine serum albumin.

In the case of an antibody which is specific for neopterin, the trihydroxypropyl side chain of neopterin must be retained as a determinant in the coupling of the hapten to the immunogenic carrier. Therefore, the simplest reaction is the alkylation, which is predominantly in the 3-position of the molecule takes place, carried out with an alkyl halide which additionally has a possibly protected, further reactive group.

For example, the reaction of neopterin with ω-bromovaleric acid methyl ester leads to the neopteryl-ω-valeric acid methyl ester, which after acidic hydrolysis of the methyl ester protecting group using the carbodiimide method on z. B. bovine serum albumin is conjugated.

Responds accordingly. B. ω-bromovaleronitrile with neopterin to 3- (4-cyanobutyl-) neopterin, which gives the corresponding imidoester by reaction in absolute methanol with hydrogen chloride, which can be reacted directly with bovine serum albumin.

Depending on the desired antibody, neopterin stands for D-erythro-Neopterin, L-erythro-Neopterin, L-threo-Neopterin or D-threo-Neopterin.

In principle, there are the same possibilities of derivatization with biopterin as with neopterin, so only the reaction with methyl 4-bromovalerate is shown as an example.

The same applies mutatis mutandis to all optical isomers L-erythro-, D-erythro-, L-threo- and D-threo-biopterin.

Specific xanthopterin antibodies are obtained by coupling the hapten to the immunogenic carrier via position 3. In order to exclude reactions at the otherwise favored positions 5 and 6, it is generally necessary to protect them temporarily, e.g. B. by methylation of the oxygen in the 6-position.

The alkylation of the nitrogen atom in the 3-position with (ω-bromovaleric acid methyl ester is facilitated by prior reaction with hexamethyldisilazane to give the O-4-trimethylsilyl derivative; otherwise the O-4-alkyl derivative is also formed. Heating in acid forms the 6- Removed methoxy and the methyl ester protecting group.

Another possibility is the link of the xanthopterin with the immunogenic carrier via the 2-amino group. Reaction with succinic anhydride in the heat gives the hemisuccinate of xanthopterin, which in the usual way using carbodiimide z. B. is conjugated to bovine serum albumin.

For the determination of reduced forms of pteridines, e.g. Antibodies can also be generated, e.g. 7,8-dihydroxanthopterin or 5,6,7,8-tetrahydrobiopterin. For this purpose, either the conjugates described above are subjected to a reduction using methods customary in pteridine chemistry, or the reactive pteridine derivatives described here are hydrogenated to the desired extent before coupling to immunogenic carriers. It should be noted, however, that the reduced pteridines, especially the 5,6,7,8-tetrahydro derivatives, are sensitive to oxygen and more sensitive to light than the oxidized forms.

In addition to being linked to the immunogenic carrier, these reactive pteridine derivatives are also used to produce the tracers necessary for immunological processes (radioactive or labeled by enzymes).

Here, for. B. the 3- (4-carboxybutyl-) D-erythro-neopterin with 3-radioiodineramine implemented according to the carbodiimide method _f , generally using ¹²⁵ J. This tracer is particularly suitable for the radioimmunological determination of neopterin.

The procedure for the production of enzyme conjugates is similar. The implementation of e.g. B. peroxidase (z. B. horseradish) and z. B. 3- (4-Carboxybutyl-) D-erythro-neopterin according to the carbodiimide method gives an enzyme conjugate which is used for the enzyme immunological determination of neopterin.

The method according to the invention is explained in more detail below with the aid of examples:

Example 1:

For the detection of malignant growth and / or viral diseases in humans, the quantitative determination of 7,8-dihydroxanthopterin in the urine by thin-layer chromatographic separation of the native urine is suitable. After the thin-layer plate has dried, a quantitative fluorometric direct evaluation takes place.

5pl human urine are applied in portions with intermediate drying using a warm air blower to a thin layer of cellulose. The solvent n-butanol / glacial acetic acid / water (20/3/7) runs 12 cm in a thin-layer chamber. After drying with a warm air blower, the thin-layer plate is scanned with a chromatogram spectral fluorometer and the fluorescence signal is recorded using a millivolt recorder. The excitation wavelength here is 378 nm, the measurement wavelength 486 nm. The fluorescence of an oxidation product, namely the Xanthopterins. The area of the band of the fluorescent spot at Rf = 27 is proportional to the 7,8-dihydroxanthopterin excreted in the urine.

Example 2:

For the detection of the Ehrlich ascites tumor and / or viral infections in the mouse, z. B. quantitative _Be - humor of 7,8-dihydro-6-hydroxylumazins in urine by hochspannungselektrophoretische separation of the native urine and subsequent fluorometric direct analysis: 10 ul on a Mausharn with Michaelis buffer (pH = 8.6 ionic strength = 0.75) impregnated sheets of paper applied in a line over a length of 3 cm. The run is done in a commercial high voltage electrophoresis chamber at 2700 volts and 45 minutes in duration. After air drying, the electrophoresis sheet is scanned 16 cm in a chromatogram fluorescence spectrophotometer, the fluorescence signal being recorded with a millivolt recorder. The excitation wavelength here is 372 nm, the measurement wavelength 456 nm. The fluorescence of an oxidation product of 7,8-dihydro-6-hydroxylumazine, namely 6-hydroxylumazine, is measured here. The area of the band at the relative electrophoretic mobility (based on 4-hydroxyhippuric acid = 100) hrM = 55 is proportional to the 7,8-dihydro-6-hydroxylumazine excreted in the urine.

Example 3:

Another possible analytical method for determining the 7,8-dihydro-6-hydroxylumazine is thin-layer chromatography on cellulose using the mobile solvent n-butanol / glacial acetic acid / water (20/3/7). The hRf value of the 7,8-dihydro-6-hydroxylumazine in this system is 25. Here, too, the 7,8-dihydro-6-hydroxylumazine is determined quantitatively by direct fluorometry at 372 nm excitation and 456 nm measurement with a chromatogram spectral fluorometer. If one excites at 356 nm and measures at 440 nm, 37 neopterin can be quantified by direct fluorometry with the hRf value.

Example 4:

7,8-Diyhdro-6-hydroxylumazine and its oxidation product 6-hydroxylumazine are simultaneously determined quantitatively by liquid chromatography. Here, 1 ml of pre-cleaned sample solution is applied to a low-pressure column 82 cm long and 6 mm inside diameter, filled with reversed-phase material Bondapak C18, particle size 37 - 75 pm. The mixture is then eluted isocratically with a 25 mM ammonium acetate solution, pH 4.5, at a flow rate of 100 ml / hour.

7,8-dihydro-6-hydroxylumazine has a retention volume of 47-53 ml and is measured by its UV absorption at 254 nm.

6-hydroxylumazine is eluted from 54 - 60 ml and measured fluorometrically at 366 nm excitation and measurement above 400 nm.

Example 5:

For the detection of malignant growth and / or viral diseases in humans, z. B. the quantitative determination of neopterin in urine by high pressure liquid chromatographic (HPLC) separation of the native urine and fluorescence detection. If necessary, a pre-separation by means of a precolumn or the like can be carried out before the chromatographic separation.

5 pl of human urine or the equivalent amount after pre-cleaning are entered into the HPLC system. The HPLC system is operated isocratically. Ammonium acetate / acetic acid (30 mol / liter, pH = 6.0) with 5% addition of methanol is used as a buffer. A 30 cm column with reversed-phase material is used as the filler. The flow rate is 2 ml / min ..

The HPLC system clearly separates the neopterin from other substances, so that the neopterin can be detected quantitatively by means of a fluorescence detector and a connected recorder. The excitation wavelength is 380 nm, the measurement wavelength 450 nm.

Simultaneously with the determination of the neopterin, the creatinine excreted in the urine is determined quantitatively, so that it can be stated in ng neopterin per mg creatinine. The reference to creatinine is always advisable when not the total daily urine but only normal urine samples are available.

Another possible analytical method for determining the neopterin consists in thin layer chromatography on cellulose with the mobile phase 5% acetic acid. The hRf value of the neopterin in this system is 65. The quantitative determination of the neopterin is carried out by direct fluorometry with 364 nm excitation and 456 nm measurement with a chromatogram spectrofluorometer.

Example 6: 6- (2-carboxy) ethoxypterin

In the dark or in dim light, 1 g of xanthopterin is heated to 70 ° C. (oil bath) with stirring in 60 ml of absolute propanediol (1,3). Then dry hydrogen chloride is introduced for about 3 h. The hydrochloride is obtained on concentration, which gives the 6- (3-hydroxy-) propyloxypterin by heating (15 min.) With water.

0.5 g of this product are treated in 20 ml of 0.1 N NaOH with KMn0 ₄ solution. The precipitate is filtered off, the filtrate provides the 6- (2-carboxy-) ethoxypterin on acidification.

Example 7: 3- (4-carboxybutyl) D-erythro-neopterin

48 mg D-erythro-neopterin are absolute in 20 ml. DMF with 50 mg of anhydrous K ₂ C0 ₃ and 0.2 ml of ω-bromovaleric acid methyl ester with exclusion of moisture at room temperature. After 18 h, a further 0.2 ml of W-bromovaleric acid methyl ester are added and the mixture is stirred for a further 50 h. Then 5 ml of 2N HCl are added until an acid reaction (pH approx. 1-2); after 5 h in H. Vak. concentrated, the residue triturated with water, suction filtered and dried.

This product can be used directly for conjugation to bovine serum albumin. The work is carried out as in Example 7 and in the following Examples 8-15 in the dark or in dim light.

Example 8: 3- (4-methoxyiminocarbonylbutyl-) D-erythro-neopterin

48 mg D _- erythro-neopterin are absolute in 10 ml. DMF with 40 mg of anhydrous K ₂ CO ₃ and 0.4 ml of ω-bromovaleronitrile with exclusion of moisture at 40 ° C (bath) stirred. After 2 days, i. H. Vak. evaporated, the residue in a little methanol. NH ₃ dissolved and separated by chromatography on silica gel. The zone corresponding to 3- (4-cyanobutyl-) D-erythro-neopterin is eluted and rotated in. 30 mg of this product are suspended in 10 ml of HCl-saturated methanol with cooling and slowly warmed to 10 ° C. with further introduction of dry HCl. The mixture then remains for 24 hours. After adding 20 ml of absolute. The product is filtered off with diethyl ether, washed and dried.

Example 9: 3- (4-carboxybutyl) L-erythro-biopterin

22 mg L-erythro-biopterin are absolute in 8 ml. DMF with 20 mg of anhydrous K ₂ CO ₃ and 0.2 ml of ω-bromovaleric acid methyl ester with exclusion of moisture for 3 days in the room temperature stirred. 6 h after the addition of 2 ml of water and 2 N HC1 to pH 1-2, i. H. Vak. evaporated, the residue treated with water, suction filtered and dried.

Example 10: 3- (4-carboxylbutyl) xanthopterin

360 mg of 6-methoxypterin are refluxed in 50 ml of hexamethyldisilazane with exclusion of moisture (20 h), after which i. Vac. constricted. The residue is absolute in 40 ml. DMF reacted with 1 ml ω-bromovaleric acid ethyl ester at 40 ° C for 30 h. The solvent is i. H. Vak. deducted. The residue is purified by chromatography on silica gel; the zone corresponding to 3- (4-carbomethoxybutyl-) 6-methoxypterin is eluted.

To remove the protective group, 100 mg of this product in 30 ml of glacial acetic acid with 3 ml of 6N HC1 are heated at 70 ° C. for 4 h. After that i. Vac. evaporated, the residue recrystallized from ethanol / water.

Example 11: Xanthopterin-N ^2- hemisuccinate

200 mg xanthopterin are heated to 70 ° C in 50 ml absolute pyridine with 250 mg succinic anhydride. After 16 h, the mixture is poured onto ice, the precipitate is filtered off with suction and washed with water. The product can be used directly for conjugation.

Example 12: 3- (Butyl-4-carboxylic acid- [3'- ¹²⁵ iodo-] tyramide) -D-erythro-neopterin

_C a. 1 m _C i 3- ¹²⁵ iodotyramine with 0.1 mg of 3- (4-carboxybutyl) D-erythro-neopterin according to Example 7 in 200 ul absolute. DMF with cooling with 80 µg dicyclohexylcarbodiimide in 50 µl absolute. Dioxane added. After standing overnight and warming to room temperature is by paper chromatography (Solvent n-propanol 1/ ₃ % aq. NH ₃ 1, v / v) the desired ¹²⁵ I-neopterin derivative is separated off and mixed with methanol. NH ₃ eluted.

Example 13: N ² - [ ³ H 7 _Ac etylxanthopterin

0.1 mg xanthopterin (0.5 µmol) are absolute in a sealed reaction vessel for 6 h with 5 m Ci [ ³ H] acetic anhydride in 200 µl. DMF heated to 100 ° C. The reaction mixture is purified by paper chromatography (mobile phase n-butanol 2/5 M acetic acid 1, v / v). The marked compound is with methanol. NH ₃ eluted.

Example 14: Xanthopterin hemisuccinate-ß-D-galactosidase conjugate

14 mg xanthopterin-N-hemisuccinate according to Example 11 and 9.2 mg tri-n-butylamine are absolute in 500 ul. DMF cooled in an ice bath, after the addition of 6.6 mg of isobutyl chloroformate, 30 min. touched. 5 mg of β-D-galactosidase in 5 ml of water are then added; the reaction is kept at pH 9 and below 10 ° C. by adding 0.1 N NaOH. After a reaction time of 5 hours, it is kept in the refrigerator overnight. The enzyme conjugate can be purified by chromatography on Sephadex G 25.

Example 15: 3- (4-carboxybutyl) D-erythro-neopterin peroxidase conjugate

17 mg of 3- (4-carboxybutyl-) D-erythro-neopterin according to Example 7 are processed according to the "mixed anhydride method" with isobutyl chloroformate in absolute. DMF implemented. The conjugation to 20 mg peroxidase (HRP) takes place in DMF / 0.5% NaHCO ₃ (1: 5), 6 h, <10 ° C. The conjugate can be purified by chromatography on Sephadex G-25.

Example 16: Enzyme immunoassay

In a small centrifuge tube (Eppendorf or Sarstedt), 100 μl serum with 400 μl PBS buffer (0.05 M phosphate pH 7.4; 0.1 M NaCl; 0.1% bovine serum albumin), 100 μl xanthopterin antiserum (rabbit 1: 1000) and 100 μl xanthopterin-β-D-galactoxidase conjugate (approx. 50 ng) incubated for 5 h at room temperature. After adding 100 μl of the second antibody (goat, anti-rabbit), the mixture is incubated for 3 h at room temperature (or overnight in the refrigerator). After centrifugation, the supernatant is suctioned off and the pellet is resuspended in 1 ml of PBS buffer. After repeating the centrifugation and suction, 0.1 ml of enzyme substrate (0.1 M phosphate pH 7.0, 2.3 mM O-nitrophenyl-β-D-galactopyranoside, 1 mM MgCl ₂ , 10 mM mercaptoethanol) is added and 2 h incubated at 23 ° C. After for standards and samples of the same time, e.g. B. 2 h, 1.5 ml of 0.2 M Na ₂ CO _{3 are} added and the optical density at 420 nm is determined.

Example 17: Radioimmunoassay

In small centrifuge tubes (Eppendorf, Sarstedt, or similar), 20 µl urine, 300 µl 0.1 M phosphate pH 7.4, 100 µl xanthopterin antiserum (rabbit, 1: 1500 with the addition of 7 µg rabbit Y globulin) and 100 µl of 2- [ ³ H-acetyl] xanthopterin (amount adjusted in the count yield of the device and the specific activity of the tracer) for 4 h at room temperature. Then 100 μl of second antibody (anti-rabbit Y-globulin serum, goat, 1:25) are added and incubated for a further 1.5 h. After adding 500 µl of water, the mixture is centrifuged and the supernatant is suctioned off. The precipitate is resuspended in 1 ml of water, centrifuged again and the supernatant is suctioned off.

The precipitate is then dissolved in 50 μl of Soluene TM 100, transferred to a scintillation vial with a total of 10 ml of Insta gel scintillation fluid (both Packard Instrument Co.) and measured in the counter (Packard).

An examination carried out according to the above examples and generally according to the method according to the invention takes up only a small amount of time. For example, an examination according to example 5 can be carried out in six minutes. The examinations according to the method according to the invention can also be repeated any number of times and thus also make it possible to monitor the course of the viral disease or to treat malignant tumors.

It depends on the pteridine in the body fluid or on the determination method used, whether the original pteridine itself is detected or its z. B. caused by light and / or air degradation product, oxidation product or reduction product.

Claims (16)

1. A method for diagnosis, in particular early detection of malignant tumors and / or viral diseases in the human or animal organism, wherein a body fluid removed from the organism is examined for the content of certain pteridines, characterized in that a preferably unconjugated, optionally derivatized 7 , 8-dihydropteridine or its oxidation product or its reduction product is qualitatively detected and / or determined semi-quantitatively or quantitatively. 2. The method according to claim 1, characterized in that preferably in the urine 7,8-Dihydroxanthopterin, 7,8-Dihydro-isoxanthopterin or 7,8-Dihydro-6-hydroxylumazine qualitatively detected and / or semi-quantitatively or quantitatively determined. 3. The method according to claim 1, characterized in that preferably in the urine or serum neopterin (6-erythro-1,2,3-trihydroxypropyl-pterine) or its hydrogenated (reduced) or substituted product qualitatively detected and / or semi-quantitative or is determined quantitatively. 4. The method according to claim 3, characterized in that D-neopterin, L-neopterin or the racemate of neopterin is detected qualitatively and / or determined semi-quantitatively or quantitatively. 5. The method according to any one of claims 1 or 2, - characterized in that the detection or the determination of the 7,8-dihydropteridine or its oxidation or reduction products under irradiation and measurement of the resulting fluorescent radiation. 6. The method according to any one of claims 1 or 2, characterized in that the quantitative determination of 7,8-dihydropteridine or. its oxidation or reduction products are carried out by high-voltage electrophoretic separation of the body fluid and subsequent fluorometric direct evaluation. 7. The method according to any one of claims 1 or 2, characterized in that the quantitative determination of 7,8-Dihydropteridinsbzw. its oxidation or reduction products are carried out by thin layer chromatography and subsequent fluorometric direct evaluation. 8. The method according to any one of claims 1 to 4, characterized in that the 7,8-dihydropteridine or its oxidation or reduction products quantitatively determined by liquid chromatography and measured fluorometrically and / or by its UV absorption. 9. The method according to any one of claims 3 or 4, characterized in that the detection of neopterin or its hydrogenated or substituted products is carried out fluorometrically by means of HPLC with reference to creatinine. 10. The method according to any one of claims 1 to 4, characterized in that the 7,8-dihydropteridine or neopterin or its oxidized, hydrogenated or substituted products are determined immunologically. 11. The method according to any one of claims 1 to 4, characterized in that the 7,8-dihydropteridine or neopterin or its oxidized, hydrogenated or substituted Products can be determined immunologically by radio immunoassay or enzyme immunoassay. 12. The method according to any one of claims 1 to 4, characterized in that the 7,8-dihydropteridine or its oxidation or reduction products are determined enzymatically. 13. The method according to any one of claims 1 to 12, characterized in that the 7,8-dihydropteridine or neopterin or its oxidized, hydrogenated or substituted products are detected or determined in a body fluid, without the prior administration of a pteridine derivative or one preparations interfering with the pteridine metabolism were removed from the organism. 14. The method according to claim 11, characterized in that antibodies are used which have been obtained by coupling an immunogenic carrier to pteridine conjugates in the 3- or 6-position of the pteridine. 15. The method according to claim 14, characterized in that tracers are used which have been obtained by coupling an enzyme or a radioactively labeled group of pteridine conjugates in the 3- or 6-position of the pteridine. 16. A diagnostic agent comprising an antibody by coupling an immuno ^g enes carrier to pteridine conjugates in the 3- or 6-position of the pteridine were obtained for the immunological detection of preferably unconjugated, optionally hydrogenated, oxidized, or derivatized 7,8-dihydropteridine and a tracer obtained by coupling an enzyme or a radiolabeled group to pteridine conjugates in the 3- or 6-position of the pteridine.